This is an Accepted Manuscript for the Microscopy and Microanalysis 2020 Proceedings. This version may be subject to change during the production process. DOI: 10.1017/S1431927620023971 1220 – Advances in Soft X-ray Tomography Venera Weinhardt 1 , Jian-Hua Chen 2 , Axel Ekman 2 , Mark A. Le Gros 1 , Gerry Mc Dermott 2 and Carolyn Larabell 1 1 Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Lawrence Berkeley National Laboratory and UCSF, Berkeley, California, United States Soft x-ray tomography (SXT) has emerged as a unique method for three-dimensional (3D) imaging of single cells [1,2]. Soft x-rays from 284 to 543 eV (2.34 to 4.4 nm) generate contrast for cells in their native, wet state without the use of chemical fixation or staining. In this so-called “water window” energy range, absorption of photons by molecular building blocks, in particular carbon and nitrogen, prevails while water is nearly transparent. Over the last decade, several groups around the world including ours designed, built and applied x-ray microscopes for 3D imaging of bacteria, yeast, algae, plankton, protozoa, viruses, and immortalized and primary cells [3,4]. This presentation will focus on describing recent advances in instrumentation, acquisition strategies, and novel application directions of SXT. Significant advances in instrumentation are necessary to accommodate the remarkable diversity of biological cells, and the imaging capabilities of SXT setups have been recently pushed to high spatial resolutions [5], switchable resolution modes [6], and alternative acquisition schemes, such as a combination of through-focus deconvolution [7,8], tomographic acquisition approaches [9] and local and half-acquisition tomography. With 5 minutes per tomogram at the Advanced Light Source (ALS), Berkeley, USA, SXT has been applied to image 3D phenotypes of yeast with thousands of cells imaged to analyze the nuclear envelope expansion in budding yeast [10]. To couple structural information with function of specific proteins, several groups have started to work on correlative imaging with confocal [11, 12] and super-resolution fluorescence imaging [13]. The example in the Figure below shows how simplified and automatic approaches are used to correlate the protein expression mapped by cryo fluorescence tomography and the 3D structure collected by SXT at the ALS of membrane-less organelles (stress granules) in osteoblast cells. As some of these organelles are suggested to consist of hierarchical subcompartments, we imaged the RNA partitioning in vitro. Beyond high-resolution visualization, soft x-ray tomography provides a quantitative measure of physico-chemical properties, such as the droplet’s viscosity, composition and size. https://doi.org/10.1017/S1431927620023971 Downloaded from https://www.cambridge.org/core. IP address: 3.238.179.243, on 17 Nov 2020 at 06:03:51, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.